In the viral vaccines field, a distinction is conventionally made between three types of viral vaccines: live virus vaccines, inactivated whole virus vaccines and subunit viral vaccines. These three types of vaccines differ by virtue of characteristics which are specific to them and which condition the composition of the excipients that are used to preserve them. Subunit vaccines containing a restricted number of viral antigens are generally easier to preserve than inactivated whole virus vaccines, for which it is sought to preserve the structural integrity of the viruses even if they are killed, or than live virus vaccines for which it is sought to also preserve the infectivity of the viruses. For each type of viral vaccine, suitable excipients have been prepared. For attenuated-virus vaccines, various stabilizing excipients have been prepared according to the preparation of attenuated viruses to be preserved. JP 57007423 describes an excipient comprising a disaccharide and on a polyalcohol in a phosphate buffer for stabilizing an attenuated strain of the measles virus. EP 065905 describes an excipient comprising one or more amino acids chosen from a group of eleven amino acids, lactose and sorbitol in a phosphate buffer for stabilizing an attenuated strain of the dengue virus. Finally, EP 0869814 describes a vaccine composition which comprises an attenuated strain of the chickenpox virus and a stabilizer containing sorbitol, mannitol, sucrose, dextran, an amino acid mixture, urea and EDTA.
In the inactivated virus vaccines field, ready-to-be administered vaccine compositions often contain proteins of animal origin, such as bovine or human albumin, gelatin or casein. Proteins are known to improve the preservation (stabilization) of these vaccines especially the vaccines that contain virus difficult to preserve. This is the case of vaccine compositions containing inactivated rabies virus. Frazatti-Gallina et al., in Vaccine (2004) Vol. 23, pp 511-517 emphasizes the importance of proteins in the stabilization of the rabies vaccine since the excipient contains albumin.
Nevertheless the presence of proteins in vaccines may represent a potential risk of disease transmission if their source is not rigorously controlled. They may also represent a potential risk of allergy, in particular when vaccines contain serum proteins, like albumin or derivatives thereof.
There therefore exists a need to find a stabilizing excipient, the composition of which does not contain protein, for stabilizing viral vaccines containing inactivated whole viruses, and in particular for stabilizing vaccines containing inactivated whole viruses that are difficult to preserve, such as rabies virus.
There also exists a need to find an excipient which is also suitable for stabilizing low-dose inactivated virus vaccines, i.e. vaccines which contain a small amount of total proteins in one effective dose of vaccine. This need is even greater for highly purified vaccines that contain very few residual protein impurities. Although the residual protein impurities can represent a potential risk of allergy, they can nevertheless contribute to stabilizing low-dose inactivated viral vaccines. They may prevent virus aggregation, virus degradation or virus adsorption that may cause a loss of efficacy of the vaccine.
To this effect, the subject of the present invention is:
A vaccine composition comprising:
a) a preparation of inactivated whole virus, and
b) a stabilizing excipient which comprises:
                i. a buffer solution,        ii. a mixture of essential and nonessential amino acids,        iii. a disaccharide,        iv. a polyol,        v. a chelating agent,        vi. urea or a urea derivative, and        vii. a nonionic surfactant.        
Preferably, the inactivated whole virus is the rabies virus.
According to one aspect of the invention, the vaccine composition is free of serum protein.
According to another aspect, the vaccine composition is free of exogenous protein of animal origin. More preferably the vaccine composition is free of exogenous product of animal origin.
In yet another aspect, the virus proteins represent at least 70% of the total proteins present in the vaccine composition.
According to yet another aspect, the total protein concentration in the vaccine composition is ≦100 μg/ml. In one particular aspect, the amount of total proteins that is contained in one effective dose of the vaccine composition is ≦100 μg.
Typically, the amount of total proteins that is contained in one effective dose of the vaccine composition is between 1 μg and 50 μg.
In another aspect of the invention, the stabilizing excipient is free of protein, or of protein and of peptide or preferably free of protein, of peptide and of oligopeptide.
According to another aspect, the mixture of essential and nonessential amino acids contained in the vaccine composition comprises at least arginine or an arginine salt and glutamic acid or a glutamic acid salt.
Preferably, the amount of essential and nonessential amino acids contained in one effective dose of the vaccine composition is between 0.5 mg and 2.5 mg.
According to another aspect, the disaccharide that is contained in the excipient is maltose.
According to yet another aspect, the polyol that is contained in the excipient is sorbitol.
Preferably, the total amount of disaccharide and of polyol contained in one effective dose of the vaccine composition is between 10 mg and 50 mg.
In another aspect, the chelating agent that is contained in the excipient is EDTA or an EDTA salt.
Preferably, the amount of chelating agent contained in one effective dose of the vaccine composition is between 0.01 mg and 0.1 mg.
Preferably, the amount of urea or of urea derivative contained in one effective dose of the vaccine composition is between 0.3 mg and 1.5 mg.
In another aspect, the nonionic surfactant that is contained in the excipient is a poloxamer.
Preferably, the poloxamer is poloxamer 188.
Preferably, the amount of nonionic surfactant contained in one effective dose of the vaccine composition is between 0.001 mg and 0.5 mg.
According to another aspect, the pH of the vaccine composition is between 7.0 and 10.0, and preferably between 7.0 and 9.0.
In another aspect, the buffer solution is a phosphate buffer, a Tris buffer, a HEPES buffer, or a mixture thereof.
In one particular aspect, the buffer solution comprises a phosphate buffer, the molarity of which is between 10 and 100 mM.
A subject of the invention is also a method for preparing a vaccine composition containing a preparation of purified inactivated whole virus, the process comprising:    a. producing a whole virus preparation by harvesting the supernatant of a virus infected cell culture.    b. purifying then inactivating the whole virus preparation is purified and then inactivated or, alternatively inactivating and then purifying the whole virus preparation,    c. diluting the preparation of purified inactivated whole virus in a stabilizing excipient, the composition of said stabilizing excipient comprising:            i. a buffer solution,        ii. a mixture of essential and nonessential amino acids,        iii. a disaccharide,        iv. a polyol,        v. a chelating agent,        vi. urea or a urea derivative, and        vii. a nonionic surfactant.        
According to one preferred embodiment, the process is carried out without introducing any exogenous product of animal origin.
In another preferred embodiment of the process, the virus is the rabies virus.
In yet another embodiment, wherein the process further comprises dividing the vaccine composition into packaging devices and optionally freeze-drying the vaccine composition
A subject of the invention is also a freeze-dried vaccine composition containing a preparation of purified inactivated whole virus, obtained according to a process of the invention.
A subject of the invention is also a stabilizing excipient for an inactivated whole virus vaccine, the composition of which comprises:                i. a buffer solution,        ii. a mixture of essential and nonessential amino acids,        iii. a disaccharide,        iv. a polyol,        v. a chelating agent,        vi. urea or a urea derivative, and        vii. a nonionic surfactant.        
Typically, the stabilizing excipient is free of protein, or of protein and of peptide, or alternatively free of protein, of peptide and of oligopeptide.
Preferably the stabilizing excipient is free of product of animal origin.